Regulation of Biosurfactant Production by Quorum Sensing in Pseudomonas fluorescens 5064, the Cause of Broccoli Head Rot Disease

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Abstract

Broccoli head rot is a destructive disease found in most broccoli production areas. The
main pathogen is the bacterium Pseudomonas fluorescens. P. fluorescens 5064, which
was first isolated from an infected broccoli head in SE Scotland, produces
biosurfactants that are important for bacterial establishment on the plant surface prior to
causing disease in broccoli.
Preliminary experiments performed in this study showed that biosurfactant production
in P. fluorescens 5064 was cell density dependent, which is a typical characteristic of
the quorum sensing mechanism. Quorum sensing is a bacterial communication
mechanism, which controls a number of key processes in growth, reproduction and
virulence via signalling molecules (quorum sensing signal) in many gram-negative
bacteria.
One aim of this study was to determine if biosurfactant production in P. fluorescens
5064 is controlled via quorum sensing. To do this, 35 surfactant-minus Tn5 mutants of
P. fluorescens 5064 were screened for their abilities to produce a quorum sensing
signal. Six of these biosurfactant-deficient mutants showed a large reduction in quorum
sensing signal production. In one mutant 6423, which contains a single Tn5 insertion,
the production of the quorum sensing signal was almost eliminated. Addition of
quorum sensing signal, either synthetic or extracted from wild type P. fluorescens 5064,
was able to restore biosurfactant production in mutant 6423. This strongly suggests that
quorum sensing regulates biosurfactant production in P. fluorescens 5064.
Attempts were made to clone and sequence the Tn5 disrupted gene in mutant 6423, but
the identity of the gene remains inconclusive.
The quorum sensing signal in wild type P. fluorescens 5064 was identified in this study
by High Pressure Liquid Chromatography and Mass Spectrometry as N-3-hydroxyoctanoyl-homoserine lactone, which has been shown by other researchers to be
present in P. fluorescens strain 2-79, but not in the strains F113, 7-14 and NCIMB
10586.
The discovery that biosurfactant production in P. fluorescens 5064 is regulated by
quorum sensing opens up a possibility for novel control of broccoli head rot. Although
only the control of biosurfactant production by quorum sensing was examined in this
study, it is possible that other virulence factors, such as pectic enzyme production, are
also controlled by quorum sensing as in other pathogenic bacteria. By blocking the
quorum sensing system, the pathogenic P. fluorescens that use this mechanism to
control virulence could potentially be rendered avirulent. In greenhouse pathogenicity
tests, a quorum sensing signal-degrading bacterium, Bacillus sp. A24, was evaluated for
biocontrol of head rot disease caused by P. fluorescens 5064 on broccoli. However, the
Bacillus sp. A24 showed only limited control effects, despite its strong quorum sensing
signal-degrading ability towards the pathogen in vitro. A subsequent test proved that
Bacillus sp. A24 is a surfactant producer itself and this could explain its ineffectiveness
in disease control.
When screening the quorum sensing signals of the 35 biosurfactant mutants, mutant
6418 was found to produce a potent antibiotic-like compound. This was identified by
thin-layer chromatography as pyrrolnitrin. Unlike wild-type P. fluorescens 5064,
mutant 6418 has lost its ability to produce virulence factors and is thus non-pathogenic.
It was therefore of interest to determine if mutant 6418 could be used as a biocontrol
agent to control broccoli head rot disease. In greenhouse pathogenicity tests, mutant
6418 significantly reduced disease by 41 %.
The practical application of this research to bacterial disease control – via the
manipulation of quorum sensing to inhibit virulence gene expression – is discussed.